Effect of nonionic surfactants [Dodecyl Maltoside and Polysorbate 20] on prevention of aggregation and conformational changes of recombinant human IFN beta_1b induced by light

Liquid protein formulations are prone to form aggregates. The effect of nonionic surfactants such as Polysorbate 20 [PS 20] and n-Dodecyl beta-D-maltoside [DDM] on the prevention of aggregation and conformational changes of recombinant human IFNbeta-1b [rhIFN beta_1b] was explored. Polysorbate has been used in formulations of protein pharmaceuticals. There have been concerns about using PS 20 due to its residual peroxide content which may negatively affect protein efficacy. n-Dodecyl beta-D-maltoside has been of interest and shown to be highly effective in prevention of aggregation. Fresh bulk of rhIFN beta_1b was formulated using DDM or different concentrations of PS 20. Formulations were exposed to light stress condition according to the ICH guideline of Q1b. The overall conformational integrity of individual samples was characterized by a combination of Circular dichroism [CD], Fluorescence spectroscopy and RP_HPLC techniques. The CD spectrum depicting the conformational integrity of rhIFN beta_1b showed 31.9% and 31.2% decreases in alpha-helix content of protein samples with 0.2% or 0.02% of PS20 compared to only18.2% of that containing 0.2% DDM. The RP-HPLC analysis also showed that the oxidized impurity in formulation containing DDM is less than those contain PS 20. Complementary analysis of the liquid formulations using IFR and UV methods also was in compliance with the data obtained by CD. Compared to PS 20, the sample of rhIFN beta_1b formulation with DDM was more resistant to the destruction effect of light. Results were in accordance with previous studies and could suggest DDM as a reliable anti-aggregation surfactant in biopharmaceutical formulations

comparison of hepatocyte cytotoxic mechanisms for docetaxel and PLGA-docetaxel nanoparticls

Docetaxel [DTX] is one of the most widely used drugs in oncology due to its high efficacy against several cancers. Though, its routine clinical administration, formulated in tween 80, causes serious side effects. Polylactide-co-glycolide [PLGA], biodegradable polyester synthesized and approved for human use, is employed to overcome these problems. In this investigation, we compare the cytotoxic mechanisms of DTX and PLGA-DTX in isolated rat hepatocytes. Cytotoxicity of DTX and PLGA-DTX were associated with reactive oxygen species formation, activation of caspases cascade, collapse of mitochondrial membrane potential [MMP], lysosomal membrane leakiness and ATP depletion. Our results also showed that CYP2E1 is involved in the oxidative stress cytotoxicity mechanism and both drugs are detoxified via phase II metabolic methylation. Furthermore, we concluded that PLGA-DTX is bioactivated by GSH. It could also potentiate hepatocyte toxicity through a mitochondrial/lysosomal toxic cross-talk. In addition to these observed differences, it is likely that mode of hepatocyte membrane penetration is different between these compounds

Preparation and antibacterial activity evaluation of 18-beta-glycyrrhetinic acid loaded PLGA nanoparticles

The aim of the present study was to formulate poly [lactide-co-glycolide] [PLGA] nanoparticles loaded with 18-beta-glycyrrhetinic acid [GLA] with appropriate physicochemical properties and antimicrobial activity. GLA loaded PLGA nanoparticles were prepared with different drug to polymer ratios, acetone contents and sonication times and the antibacterial activity of the developed nanoparticles was examined against different gram-negative and gram-positive bacteria. The antibacterial effect was studied using serial dilution technique to determine the minimum inhibitory concentration of nanoparticles. Results demonstrated that physicochemical properties of nanoparticles were affected by the above mentioned parameters where nanoscale size particles ranging from 175 to 212 nm were achieved. The highest encapsulation efficiency [53.2 +/- 2.4%] was obtained when the ratio of drug to polymer was 1:4. Zeta potential of the developed nanoparticles was fairly negative [-11 +/- 1.5]. In-vitro release profile of nanoparticles showed two phases: an initial phase of burst release for 10 h followed by a slow release pattern up to the end. The antimicrobial results revealed that the nanoparticles were more effective than pure GLA against P. aeuroginosa, S. aureus and S. epidermidis. This improvement in antibacterial activity of GLA loaded nanoparticles when compared to pure GLA may be related to higher nanoparticles penetration into infected cells and a higher amount of GLA delivery in its site of action. Herein, it was shown that GLA loaded PLGA nanoparticles displayed appropriate physicochemical properties as well as an improved antimicrobial effect

Pasteurization of IgM-enriched immunoglobulin

Human plasma proteins are important for therapy or prophylaxis of human diseases. Due to the preparation of human plasma proteins from human plasma pools and risk of contamination with human viruses, different viral reduction treatments such as: pasteurization, solvent/detergent, dry heat treatment, steam treatment, beta-propiolactone/UV and nanofiltration have been implemented. As pasteurization can be performed for liquid protein, this method [a 10-hour heat treatment of the aqueous solutions at 60°C] was introduced into the manufacturing procedure of IgM-enriched immunoglobulin, to improve its safety further. The efficiency of this method for inactivation of viruses was evaluated by the use of Foot-and-Mouth Disease Virus [a non-enveloped virus] and Infectious Bovine Rhinotracheitis [IBR] Virus [a lipid-enveloped virus]. Pasteurization inactivated Foot-and-Mouth Disease Virus by 7 log10 and for IBR Virus by 5log10. These findings show a significant added measure of virus safety associated with pasteurization of IgM-enriched immunoglobulin preparation

Preparation of enriched immunoglobulin M and immunoglobin A from human plasma

As IgM and IgA-enriched preparations are needed to complete the immunotherapeutic spectrum, a simple procedure is described for the preparation of IgM and IgA-enriched immunoglobulins. Fraction III which was prepared by cold ethanol fractionation was treated by octanoic acid followed by ethanol precipitation and ion-exchange chromatography using Sephadex DEAE A-50 and 0.1 M tris-0.35M NaC1 buffer, pH 8.1, resulting in recovery of 85% IgM, 84% IgA and 33% IgG. The comparison of our results with immunoglobulins' percentage in plasma indicates that IgM and IgA-enrichment was obtained by three times

Preparation of ethylcellulose coated gelatin microspheres as a multiparticulate colonic delivery system for 5-aminosalicilic acid

In the long-term management of ulcerative colitis patients, repeat dosing maybe required. Since 5-ASA is largely absorbed from the upper intestine, selective delivery of drugs into the colon may be regarded as a better method of drug delivery with fewer side effects and a higher efficacy. The aim of this study was to prepare and evaluate a double coated multiparticulate system for 5-ASA delivery using gelatin and ethylcellulose as the primary and secondary polymer respectively. Gelatin microspheres containing 5-aminosalicylic acid was produced using the solvent evaporation method. Prepared gelatin microspheres were spherical, free flowing, non-aggregated and showed no degradation in the acidic medium. Entrapment efficacy of microspheres was about 50%. Results showed that drug release was fast and complete and is affected by the amount of core material entrapped. Gelatin microspheres were then coated by ethylcellulose using a coacervation phase separation technique. The idea for this approach was to prepare a delayed drug delivery system, in which, ethylcellulose protects particles for the first 6 h transit through the gastrointestinal tract. However, it was shown that this system could provide a suitable drug release pattern for colonic delivery of active agents, as 30% of the drug was released from the ethylcellulose-coated microcapsules within 6 h, while this amount was 90% of the loaded drug for gelatin microspheres under the same condition

Solvent-detergent treatment of IgM-enriched immunoglobulin

Viral safety of human plasma products plays a key role in their safe uses. Solvent- detergent [SD] virus-inactivation method has gained widespread popularity in the manufacture of biological products. This treatment which inactivates lipid-enveloped viruses effectively consists of incubation of a plasma protein solution in the presence of a non-volatile organic solvent and a detergent. In this study, IgM-enriched immunoglobulin was incubated at 24 °C for 6 h under slow stirring in the presence of tri[n-butyl] phosphate [0.3% w/w] as solvent and tween 80 [1% w/w] as detergent. After completion of the inactivation process and removal of the solvent-detergent, the ability of SD-treatment to remove Infectious Bovine Rhinotracheitis [IBR] virus [a lipid-enveloped virus] and Foot-and-Mouth Disease virus [a non-enveloped virus] were evaluated by "virus spiking studies" using a scaled down process. Reduction factor of 4 log was obtained for the SD-treatment of IgM-enriched immunoglobulin spiked with IBR virus. No virus inactivation was observed in the SD-treated IgM-enriched immunoglobulin, spiked with Foot-and-Mouth Disease virus. It was concluded that treatment of IgM-enriched immunoglobulin with TNBP-TWEEN 80 may be considered as an efficient lipid-enveloped virus inactivation step in the manufacture of this product

effect of solvent and crystallization conditions on habit modification of carbamazepine

Physical characteristics of carbamazepine crystals grown from pure ethanol or acetone under different conditions were studied for the morphology of crystals by scanning electron microscope, x-ray powder diffraction and FT-IR, and for thermodynamic properties by differential scanning calorimeter. Also the dissolution behavior and compaction properties of crystals were studied. The results showed that crystallization of carbamazepine using watering-out method produced needle shape crystals while by the other methods such as reducing temperature or solvent evaporation produced polyhedral crystals in alcohol and thin plate-like crystals in acetone. The crystals which were grown from acetone were larger than those from alcohol. Differential scanning calorimetery and x-ray powder diffraction showed no evidence of poly-morphism for carbamazepine crystallized by reducing the temperature or by the solvent evaporation in contrast with the crystals produced by the watering out technique. Crystallization of carbamazepine by different methods especially watering-out technique improved its dissolution rate and compactibility and produced high crushing strength compacts without capping

Effects of formulation variables on nifedipine microspheres prepared by solvent evaporation technique

Preparation and characterization of nifedipine microspheres using ethylcellulose as matrix polymer is described. Nifedipine microspheres were prepared by solvent evaporation technique. The influence of different parameters such as the effect of the concentration of internal and external phases, the amount of drug and the rate of stirring of the medium on the size distribution of microspheres were studied. The effect of drug/polymer ratio and mean particle size on the drug release pattern were also evaluated. Drug release from nifedipine microspheres was studied in a medium, which simulated the change in pH of the pathway of the microspheres from stomach to intestine. It was found that with increase in the concentration of the internal phase, the size of microspheres became larger. Increasing the amount of polyvinyl alcohol in the external phase reduced the size of microspheres. Dissolution was found to be inversely related to the pH, in a way that drug release decreased at higher pH: Drug release from microspheres with small mean particle size was faster than those with large mesh particle size and followed Higuchi model of kinetics